1. Field of the Invention
The present invention relates to a method for producing a contrast agent.
2. Description of the Related Art
Fluorescent imaging and photoacoustic imaging have received attention as optical imaging methods capable of noninvasive diagnosis in recent years.
Fluorescent imaging is a method of detecting fluorescence emitted from a fluorescent dye by irradiating the fluorescent dye with light and is broadly used. Photoacoustic imaging is a method of imaging an object by irradiating the object with light, and detecting the intensity of acoustic waves generated by volume expansion resulting from the emission of heat from the molecules of the irradiated object and the position where the acoustic waves have been generated. In the fluorescent imaging and photoacoustic imaging, a dye may be used as a contrast agent for increasing the intensity of fluorescence or acoustic waves from a targeted site.
PCT Japanese Translation Patent Publication No. 2012-520856 discloses a contrast agent used for photoacoustic imaging and optical imaging and containing a conjugate of a cyanine dye and a polyethylene glycol (hereinafter may be abbreviated as PEG). In order to form the conjugate, Cy7-NHS (N-hydroxysuccinimide) ester (formula (1)), which is a cyanine dye difficult to form aggregates in water, is combined with a PEG containing amino groups at both ends in water. Then, the reaction product is purified by reversed phase chromatography using an aqueous solution.

If a cyanine dye containing a hydrophobic portion such as a naphthyl group is combined with a PEG containing an amino group, the cyanine dye is required to have a functional group capable of reacting with the amino group, such as carboxy or N-hydroxysuccinimide ester.
While N-hydroxysuccinimide esters can react with the amino group without using a condensing agent, the reactivity of the N-hydroxysuccinimide esters decreases with time. From the viewpoint of maintaining the reactivity of the cyanine dye with the amino group, therefore, a cyanine dye containing a carboxyl group is advantageously used.
Unfortunately, conjugates of a cyanine dye containing a carboxyl group and partially having a hydrophobic structure such as the naphthyl group and a PEG containing an amino group are difficult to purify by reversed phase chromatography using water or the like because the particles of the dye aggregate together in the column.
In order to increase the dispersibility of the dye in water, a sulfo or sulfonate group may be introduced to change the structure of the dye. However, this can vary reactivity between the carboxyl group and the amino group and light emitting characteristics. Accordingly, it is desired to synthesize a conjugate using a cyanine dye tending to form aggregates in water without changing the structure of the cyanine dye so as to increase the dispersibility, and purify the conjugate.